Bacillus anthracis is an endospore-forming zoonotic bacterium of significant concern as a bioterrorism agent. The route by which the spore gains access to a human host defines the nature of the resulting disease, cutaneous, gastrointestinal, or pulmonary. Gastrointestinal and pulmonary anthrax are associated with high mortality rates. The composition of the B. anthracis spore, especially its interspace and exosporium layers, is incompletely understood. Enzymes, including alanine racemase, inosine preferring nucleoside hydrolase, and nitric oxide synthase, are found in the outer spore coat, interspace, and exosporium layers. These enzymes are thought to function in spore germination and defense against the host innate immune system. We have identified the presence of proteins associated with the regulation of cyclic di-GMP levels in spores of B. anthracis. Cyclic di-GMP is an important regulator that has been identified in a number of bacterial pathogens. It is a critical regulator of a number of virulence attributes including biofilm formation, motility, intracellular survival, expression of surface adhesions, expression of pili, resistance to oxidative stress, and modulation of the host immune response. A role for this cyclic nucleotide regulator in spore biology, either sporulation or germination, has not been reported. We propose to study GGDEF domain-containing proteins whose expression profile and/or protein fusion studies indicate that they are spore proteins. Initial studies with one of these determinants have shown an effect on spore maturation. In this project, we will determine the location within the spores for these GGDEF proteins, the effects on spore structure and function associated with loss of these proteins both singly and in combination, and the effects on spore biology in macrophage cell cultures and a mouse model of infection. The goals of these experiments are to determine the roles of the GGDEF proteins on sporulation, germination, and virulence. Because the spore is the infectious form of this zoonotic pathogen, this information is important to our understanding of the B. anthracis infectious process and may identify potential targets for therapeutic intervention against the initial steps in B. anthracis infections. Information obtained from this study may have broader applicability to understanding the infectious process of other endospore-forming bacterial pathogens.